studies

658A AASLD ABSTRACTS HEPATOLOGY, October, 2015
fourth week and twice at 1-week intervals to investigate their
importance in the pathogenesis. Functional effects of Tregs on
lipid metabolism, oxidative stress, and macrophage activation
were then examined. Results: Chronic alcohol feeding induced
liver steatosis and increased serum alanine aminotransferase. A
decrease of hepatice Tregs was observed in these mice. Adaptive
transfer of Tregs was able to abolish hepatic inflammation
and liver injury. Adaptive transfer of Tregs downregulated the
enhanced expression of nuclear Sterol regulatory element-binding
protein 1c (SREBP1c) and its downstream genes induced
in alcohol-fed mice. This intervention also ameliorated hepatic
oxidative stress, increased the level of phosphorylatied adenosine
monophosphate-activated protein kinase (AMPK), peroxisome
proliferator-activated receptor (PPAR) α in the nucleus,
and its downstream genes involved in fatty acid metabolism,
which were inhibited by alcohol-feeding. Furthermore, Tregs
inhibited MCP-1 and TNF- overproduction and macrophage
activation in the liver. In vitro, Tregs suppressed the expression
of MCP-1, TNFα, and CD14 on monocytes/macrophages that
underwent LPS and alcohol co-treatment. This suppression was
markedly abrogated by neutralizing anti-IL-10 mAbs. Conclusions:
Our results indicate that Tregs suppress the development
of AFL, in part through modulating lipid metabolism, oxidative
stress, and the macrophage pro-inflammatory response.
Disclosures:
Qin Ning - Advisory Committees or Review Panels: ROCHE, NOVARTIS, BMS,
MSD, GSK; Consulting: ROCHE, NOVARTIS, BMS, MSD, GSK; Grant/Research
Support: ROCHE, NOVARTIS, BMS; Speaking and Teaching: ROCHE, NOVAR-
TIS, BMS, MSD, GSK
The following authors have nothing to disclose: Hongwu Wang, Ting Wu, Yaqi
Wang, Xiaojing Wang, Xiaoping Luo
910
NOX2 deficiency attenuates ethanol-induced hepatic
steatosis by down-regulating CB1R-mediated lipogenesis
in mice
Jong-Min Jeong, So Yeon Kim, Won-IL Jeong; KAIST, Daejeon,
Korea (the Republic of)
Background: Lines of evidence have suggested that alcohol-mediated
oxidative stress plays important roles in hepatic
steatosis, in which hepatic stellate cells (HSCs) stimulate CB1
receptor (CB1R)-mediated hepatic lipogenesis by producing
an endocannabinoid, 2-arachidonoylglycerol (2-AG). However,
it is not clear yet whether oxidative stress has an effect
on endocannabinoid/CB1R signaling pathway. In the present
study, we investigated the role of NADPH oxidase 2 (NOX2) in
hepatic CB1R signaling pathway during alcoholic steatosis in
mice. Methods: WT and NOX2 deficient (NOX2 -/- ) mice were
fed with liquid ethanol diet for 8 weeks. Sera and liver tissues
of mice were used for biochemical analyses, hitopathological
observation, fluorescence-activated sorting (FACS) analysis
and real-time PCR analyses. LPS treatment, NOX2 siRNA, measurement
of reactive oxygen species (ROS), immunoblotting
and immunostaining were performed in primary hepatic macrophages
and Raw 264.7 cell lines. Results: In histological findings,
livers of NOX2 -/- mice showed less hepatic steatosis than
WT mice. In FACS analyses, populations of Gr1 + CD11b + and
F4/80 + CD11b + cells in WT mice were similar with those of
NOX2 -/- mice. However, gene expression of inflammatory cytokines
in WT mice was significantly higher than that of NOX2 -
/-
mice. In addition, LPS-mediated ROS generation in primary
hepatic and Raw 264.7 macrophages was remarkably attenuated
as NOX2 and toll-like receptor 4 (TLR4) were depleted in
macrophages, suggesting that TLR4 activation was implicated
in NOX2-mediated ROS generation. Furthermore, in co-culturing
macrophages with HSCs, we found that NOX2-mediated
ROS generation in hepatic macrophages increased gene
expression of diacylglycerol lipases, specific enzymes for 2-AG
production, in HSCs, leading to enhance hepatic lipogenesis
through CB1R signaling in hepatocyte. Conclusions: Based on
our data, LPS-mediated NOX2 activation in hepatic macrophage
played a decisive role on alcoholic hepatic steatosis by
stimulating endocannabinoid production of HSCs and CB1R
signaling in hepatocyte. Therefore, NOX2 might be a therapeutic
candidate for alcoholic liver disease.
Disclosures:
The following authors have nothing to disclose: Jong-Min Jeong, So Yeon Kim,
Won-IL Jeong
911
L-Fabp deletion attenuates both hepatic steatosis and
fibrosis resulting from hepatic microsomal triglyceride
transfer protein (Mttp) deletion
Elizabeth P. Newberry 2 , Susan M. Kennedy 2 , Yan Xie 2 , Hui
Jiang 2 , Anping Chen 1 , Daniel S. Ory 2 , Nicholas O. Davidson 2 ;
1 St. Louis University, St Louis, MO; 2 Washington University School
of Medicine, St Louis, MO
Genetic or pharmacological inhibition of hepatic microsomal
triglyceride transfer protein (Mttp) blocks VLDL secretion
causing hepatic steatosis. However, the effects on fibrosis are
unknown. Here we asked if liver specific (Alb Cre) Mttp deletion
(Mttp-LKO) promotes hepatic stellate cell activation and
fibrosis and if this phenotype is altered by germline L-Fabp
deletion (DKO mice). Mttp-LKO mice fed low fat chow showed
~10 fold increased hepatic triglyceride (TG) vs C57BL/6J controls
at 12 weeks, with upregulated expression of fibrotic genes
(Col1a1, αSMA, Ctgf) and increased collagen staining (C57,
4.1 ± 0.6 foci/field; Mttp-LKO, 10.1 ± 0.9, n=5-7/group).
Steatosis was significantly attenuated in DKO mice (223 ± 13
mg TG/mg protein vs 331 ± 36, Mttp-LKO, n=6), with reduced
expression of fibrogenic mRNAs and decreased fibrotic staining
(DKO, 5.1 ± 0.5 foci/field). High fat diet fed Mttp-LKO
mice also exhibited 2.5-fold increased hepatic TG, which
was attenuated in DKO mice, again with attenuated Col1a1
gene expression. Expression of lipid droplet proteins Pln2 and
Cidec mRNA were reduced in DKO vs Mttp-LKO, consistent
with reduced steatosis. In vitro FA trafficking revealed ~25%
decreased incorporation of FA into TG in DKO hepatocytes,
yet reduced FA oxidation compared to Mttp-LKO, suggesting
altered FA compartmentalization. ER stress response (Xbp1s,
Atf4, Ern1) was decreased ~40% in DKO livers vs both C57
and Mttp-LKO. Oxidative stress, assessed by lipoperoxide levels,
was increased 4-fold in Mttp-LKO vs DKO (Mttp-LKO, 6.8
± 0.9 nmol LPO/mg protein; DKO, 1.7 ± 0.3, n=4). To understand
the basis for reduced fibrosis in mice lacking L-Fabp, we
undertook broad range lipidomic analyses in livers of Mttp-
LKO and DKO mice. As expected, TG mass was decreased
~25% in DKO, with a disproportionate reduction in TG species
containing essential fatty acids C18:2 and C18:3 (Mttp-LKO,
11.9 ± 0.6; DKO, 8.0 ± 0.6, n=4-5/group, p